PRESENTATION OF A PCR-NUCLEASE PROTECTION STRATEGY FOR MINIMAL RESIDUAL DISEASE MONITORING IN B-ALL

Methods for detecting residual malignant cells in patients suffering f rom lymphoid malignancies have neither been sufficiently sensitive nor easy to routinize, hampering a potential prediction of disease outcom e. Taking advantage of clone-specific DNA sequences, generated during lymphocyte differentiation and the polymerase chain reaction, some str ategies have been developed for several groups. Up to now the most spe cific and sensitive methodology, which consists of designing leukemia- specific oligonucleotides, requires sequencing of the complementary de termining region III-DNA for each particular patient and is too labori ous to be applied to each case for routine monitoring in most hospital laboratories. In an attempt to achieve an easy way to detect residual malignant cells in B lymphoproliferative diseases, we have used a new PCR-based approach, named here as PCR-nuclease protection assay, cons isting of: (i) amplification of DNA segments corresponding to the comp lementarity determining region III of the immunoglobulin heavy chain g enes from samples at disease diagnosis; (ii) isolation of the disease- specific single-stranded DNA; (iii) labeling of the single-stranded DN A to generate specific probes; (iv) hybridization to amplified DNA fro m samples corresponding to different disease phases; and (v) digestion with S1-nuclease. Using this approach, we could detect one malignant cell in a background of 10(5) healthy cells. The sensitivity and speci ficity of this approach compares with those of the above mentioned spe cific oligonucleotide strategy in detecting residual malignant B cells . Moreover, this strategy is much less tedious and could be used by mo st hospital laboratories.